MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

MC1R, a G-protein coupled receptor, triggers ultraviolet light-induced melanin synthesis and DNA repair in melanocytes and is implicated in the pathogenesis of melanoma. Although widely expressed in different tissue types, its function in non-cutaneous tissue is relatively unknown. Herein, we demonstrate that disruptive MC1R variants associated with melanomagenesis are less frequently found in patients with several cancers. Further exploration revealed that breast cancer tissue shows a significantly higher MC1R expression than normal breast tissue, and knocking down MC1R significantly reduced cell proliferation in vitro and in vivo. Mechanistically, MC1R signaling through the MC1R-cAMP-CREB/ATF-1 and MC1R-ERK-NFκB axes accelerated the G1-S transition in breast cancer cells. Our results revealed a new association between MC1R and breast cancer, which could be potentially targeted therapeutically. Moreover, our results suggest that MC1R-enhancing/activating therapies should be used cautiously, as they might be pro-tumorigenic in certain contexts.

SARS-CoV-2 3CL-protease inhibitors derived from ML300: investigation of P1 and replacements of the 1,2,3-benzotriazole.

Starting from compound 5 (CCF0058981), a structure-based optimization of the P1 subsite was performed against the severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CLpro). Inhibitor 5 and the compounds disclosed bind to 3CLpro using a non-covalent mode of action that utilize a His163 H-bond interaction in the S1 subpocket. In an effort to examine more structurally diverse P1 groups a number of azoles and heterocycles were designed. Several azole ring systems and replacements, including C-linked azoles, with similar or enhanced potency relative to 5 were discovered (28, 29, and 30) with demonstrated IC50 values less than 100 nM. In addition, pyridyl and isoquinoline P1 groups were successful as P1 replacements leading to 3-methyl pyridyl 36 (IC50 = 85 nM) and isoquinoline 27 (IC50 = 26 nM). High resolution X-ray crystal structures of these inhibitors were utilized to confirm binding orientation and guide optimization. These findings have implications towards antiviral development and preparedness to combat SARS-like zoonotic coronavirus outbreaks.

Redefining the PTEN promoter: identification of novel upstream transcription start regions.

Germline mutation of PTEN is causally observed in Cowden syndrome (CS) and is one of the most common, penetrant risk genes for autism spectrum disorder (ASD). However, the majority of individuals who present with CS-like clinical features are PTEN-mutation negative. Reassessment of PTEN promoter regulation may help explain abnormal PTEN dosage, as only the minimal promoter and coding regions are currently included in diagnostic PTEN mutation analysis. Therefore, we reanalyzed the architecture of the PTEN promoter using next-generation sequencing datasets. Specifically, run-on sequencing assays identified two additional transcription start regions (TSRs) at -2053 and -1906 basepairs from the canonical start of PTEN, thus extending the PTEN 5'UTR and redefining the PTEN promoter. We show that these novel upstream TSRs are active in cancer cell lines, human cancer and normal tissue. Furthermore, these TSRs can produce novel PTEN transcripts due to the introduction of new splice donors at -2041, -1826 and -1355, which may allow for splicing out of the PTEN 5'UTR or the first and second exon in upstream-initiated transcripts. Combining ENCODE ChIP-seq and pertinent literature, we also compile and analyze all transcription factors (TFs) binding at the redefined PTEN locus. Enrichment analyses suggest that TFs bind specifically to the upstream TSRs may be implicated in inflammatory processes. Altogether, these data redefine the architecture of the PTEN promoter, an important step toward a comprehensive model of PTEN transcription regulation, a basis for future investigations into the new promoters' role in disease pathogenesis.

PTEN interacts with RNA polymerase II to dephosphorylate polymerase II C-terminal domain.

Gene transcription is a highly complex and strictly regulated process. RNA polymerase II (Pol II) C-terminal domain (CTD) undergoes massive cycles of phosphorylation and dephosphorylation during the process of gene transcription. These post-translational modifications of CTD provide an interactive platform for various factors required for transcription initiation, elongation, termination, and co-transcriptional RNA processing. Pol II CTD kinases and phosphatases are key regulators and any deviation may cause genome-wide transcriptional dysregulation leading to various pathological conditions including cancer. PTEN, a well known tumor suppressor, is one of the most commonly somatically altered in diverse malignancies. When mutated in the germline, PTEN causes cancer predisposition. Numerous studies have demonstrated that PTEN regulates the expression of hundreds of genes, however, no mechanism is known so far. PTEN is a dual specificity phosphatase, using both lipid and protein as substrates. In the present study, we demonstrate that PTEN interacts with the RNA Pol II and that PTEN expression is inversely correlated with global phosphorylation of Pol II CTD. Furthermore, PTEN dephosphorylates Pol II CTD in vitro with a significant specificity for Ser5p. Interestingly, ChIP-seq data analysis revealed that PTEN globally binds to promoter proximal regions, and PTEN loss increases genome-wide Pol II Ser5p occupancy, suggest that PTEN is a Pol II CTD phosphatase. Our observations demonstrate an unexplored function of PTEN with the potential of global transcriptional regulation, adding a new dimension to somatic carcinogenesis and germline cancer predisposition.

PTEN modulates gene transcription by redistributing genome-wide RNA polymerase II occupancy.

Control of gene expression is one of the most complex yet continuous physiological processes impacting cellular homeostasis. RNA polymerase II (Pol II) transcription is tightly regulated at promoter-proximal regions by intricate dynamic processes including Pol II pausing, release into elongation and premature termination. Pol II pausing is a phenomenon where Pol II complex pauses within 30-60 nucleotides after initiating the transcription. Negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) contribute in the establishment of Pol II pausing, and positive transcription elongation factor b releases (P-TEFb) paused complex after phosphorylating DSIF that leads to dissociation of NELF. Pol II pausing is observed in most expressed genes across the metazoan. The precise role of Pol II pausing is not well understood; however, it's required for integration of signals for gene regulation. In the present study, we investigated the role of phosphatase and tensin homolog (PTEN) in genome-wide transcriptional regulation using PTEN overexpression and PTEN knock-down models. Here we identify that PTEN alters the expression of hundreds of genes, and its restoration establishes genome-wide Pol II promoter-proximal pausing in PTEN null cells. Furthermore, PTEN re-distributes Pol II occupancy across the genome and possibly impacts Pol II pause duration, release and elongation rate in order to enable precise gene regulation at the genome-wide scale. Our observations demonstrate an imperative role of PTEN in global transcriptional regulation that will provide a new direction to understand PTEN-associated pathologies and its management.

Characterization of cryptic splicing in germline PTEN intronic variants in Cowden syndrome.

Germline mutations in the tumor-suppressor gene PTEN predispose to subsets of Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome, and autism. Evidence-based classification of PTEN variants as either deleterious or benign is urgently needed for accurate molecular diagnosis and gene-informed genetic counseling. We studied 34 different germline PTEN intronic variants from 61 CS patients, characterized their PTEN mRNA processing, and analyzed PTEN expression and downstream readouts of P-AKT and P-ERK1/2. While we found that many mutations near splice junctions result in exon skipping, we also identified the presence of cryptic splicing that resulted in premature termination or a shift in isoform usage. PTEN protein expression is significantly lower in the group with splicing changes while P-AKT, but not P-ERK1/2, is significantly increased. Our observations of these PTEN intronic variants should contribute to the determination of pathogenicity of PTEN intronic variants and aid in genetic counseling.

Germline compound heterozygous poly-glutamine deletion in USF3 may be involved in predisposition to heritable and sporadic epithelial thyroid carcinoma.

Cowden syndrome (CS) is an autosomal dominant disorder that predisposes to breast, thyroid, and other epithelial cancers. Differentiated thyroid carcinoma (DTC), as one of the major component cancers of CS, is the fastest rising incident cancer in the USA, and the most familial of all solid tumours. To identify additional candidate genes of CS and potentially DTC, we analysed a multi-generation CS-like family with papillary thyroid cancer (PTC), applying a combined linkage-based and whole-genome sequencing strategy and identified an in-frame germline compound heterozygous deletion, p.[Gln1478del];[Gln1476-Gln1478del] in USF3 (previously known as KIAA2018). Among 90 unrelated CS/CS-like individuals, 29% were found to have p.[Gln1478del];[Gln1476-Gln1478del]. Of 497 TCGA PTC individuals, 138 (27%) were found to carry this germline compound deletion, with somatically decreased tumour USF3 expression. We demonstrate an increased migration phenotype along with enhanced epithelial-to-mesenchymal transition (EMT) signature after USF3 knockdown or USF3 p.[Gln1478del];[Gln1476-Gln1478del] overexpression, which sensitizes cells to the endoplasmic reticulum (ER) stress. Loss of USF3 function induced cell necrosis-like features and impaired respiratory capacity while providing a glutamine-dependent cell survival advantage, strongly suggests a metabolic survival and migration-favouring microenvironment for carcinogenesis. Therefore, USF3 may be involved in the predisposition of thyroid cancer. Importantly, the results that glutamine-dependent survival and sensitivity to ER stress in USF3-deficient cells provide avenues for therapeutic and adjunct preventive interventions for both sporadic cancer as well as cancer predisposition syndromes with similar mechanisms.

Erratum to: Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

[This corrects the article DOI: 10.1186/s13229-015-0056-6.].

Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

BACKGROUND: Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impairment in social communication/interaction and inflexible/repetitive behavior. Several lines of evidence support genetic factors as a predominant cause of ASD. Among those autism susceptibility genes that have been identified, the PTEN tumor suppressor gene, initially identified as predisposing to Cowden heritable cancer syndrome, was found to be mutated in a subset of ASD patients with extreme macrocephaly. However, the ASD-relevant molecular mechanism mediating the effect of PTEN mutations remains elusive. METHODS: We developed a Pten knock-in murine model to study the effects of Pten germline mutations, specifically altering subcellular localization, in ASD. Proteins were isolated from the hemispheres of the male littermates, and Western blots were performed to determine protein expression levels of tyrosine hydroxylase (TH). Immunohistochemical stains were carried out to validate the localization of TH and dopamine D2 receptors (D2R). PC12 cells ectopically expressing either wild-type or missense mutant PTEN were then compared for the differences in TH expression. RESULTS: Mice carrying Pten mutations have high TH and D2R in the striatum and prefrontal cortex. They also have increased phosphorylation of cAMP response element-binding protein (CREB) and TH. Mechanistically, PTEN downregulates TH production in PC12 cells via inhibiting the phosphoinositide 3-kinase (PI3K)/CREB signaling pathway, while PTEN reduces TH phosphorylation via suppressing MAPK pathway. Unlike wild-type PTEN but similar to the mouse knock-in mutant Pten, three naturally occurring missense mutations of PTEN that we previously identified in ASD patients, H93R, F241S, and D252G, were not able to suppress TH when overexpressed in PC12 cells. In addition, two other PTEN missense mutations, C124S (pan phosphatase dead) and G129E (lipid phosphatase dead), failed to suppress TH when ectopically expressed in PC12 cells. CONCLUSIONS: Our data reveal a non-canonical PTEN-TH pathway in the brain that may work as a core regulator of dopamine signaling, which when dysfunctional is pathogenic in ASD.

Exome Sequencing Reveals Germline SMAD9 Mutation That Reduces Phosphatase and Tensin Homolog Expression and Is Associated With Hamartomatous Polyposis and Gastrointestinal Ganglioneuromas.

Hamartomatous polyposis syndromes (HPS) account for a small but appreciable proportion of inherited gastrointestinal cancer predisposition syndromes; patients with HPS have an increased risk for colon and extracolonic malignancies. We present a unique case of familial juvenile polyposis syndrome associated with gastrointestinal ganglioneuromas of unknown etiology. The patient was tested for HPS-associated genes, but no mutation was detected. Exome sequencing identified a germline heterozygous mutation in SMAD9 (SMAD9(V90M)). This mutation was predicted to be an activating mutation. HEK cells transfected to express SMAD9(V90M) had reduced expression of phosphatase and tensin homolog; this reduction was also observed in a polyp from the patient. We have therefore identified a new susceptibility locus for HPS. Patients with hamartomatous polyposis in the colon associated with ganglioneuromatosis should be referred for genetic assessments.

Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production.

PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal-dominant genetic condition underlying a subset of autism spectrum disorder (ASD) with macrocephaly. Caused by germline mutations in PTEN, PHTS also causes increased risks of multiple cancers via dysregulation of the PI3K and MAPK signaling pathways. Conditional knockout models have shown that neural Pten regulates social behavior, proliferation and cell size. Although much is known about how the intracellular localization of PTEN regulates signaling in cancer cell lines, we know little of how PTEN localization influences normal brain physiology and behavior. To address this, we generated a germline knock-in mouse model of cytoplasm-predominant Pten and characterized its behavioral and cellular phenotypes. The homozygous Pten(m3m4) mice have decreased total Pten levels including a specific drop in nuclear Pten and exhibit region-specific increases in brain weight. The Pten(m3m4) model displays sex-specific increases in social motivation, poor balance and normal recognition memory-a profile reminiscent of some individuals with high functioning ASD. The cytoplasm-predominant protein caused cellular hypertrophy limited to the soma and led to increased NG2 cell proliferation and accumulation of glia. The animals also exhibit significant astrogliosis and microglial activation, indicating a neuroinflammatory phenotype. At the signaling level, Pten(m3m4) mice show brain region-specific differences in Akt activation. These results demonstrate that differing alterations to the same autism-linked gene can cause distinct behavioral profiles. The Pten(m3m4) model is the first murine model of inappropriately elevated social motivation in the context of normal cognition and may expand the range of autism-related behaviors replicated in animal models.

Activation of AR sensitizes breast carcinomas to NVP-BEZ235's therapeutic effect mediated by PTEN and KLLN upregulation.

NVP-BEZ235 is a newly developed dual PI3K/mTOR inhibitor, being tested in multiple clinical trials, including breast cancer. NVP-BEZ235 selectively induces cell growth inhibition in a subset, but not all, breast cancer cell lines. However, it remains a challenge to distinguish between sensitive and resistant tumors, particularly in the pretreatment setting. Here, we used ten breast cancer cell lines to compare NVP-BEZ235 sensitivity and in the context of androgen receptor (AR) activation during NVP-BEZ235 treatment. We also used female SCID mice bearing breast tumor xenografts to investigate the beneficial effect of dihydrotestosterone/NVP-BEZ235 combination treatment compared with each alone. We found that AR-positive breast cancer cell lines are much more sensitive to NVP-BEZ235 compared with AR-negative cells, regardless of PTEN or PI3KCA status. Reintroducing AR expression in NVP-BEZ235 nonresponsive AR-negative cells restored the response. DHT/NVP-BEZ235 combination not only resulted in a more significant growth inhibition than either drug alone, but also achieved tumor regression and complete responses for AR(+)/ER(+) tumors. This beneficial effect was mediated by dihydrotestosterone (DHT)-induced PTEN and KLLN expression. Furthermore, DHT could also reverse NVP-BEZ235-induced side effects such as skin rash and weight loss. Our data suggest that AR expression may be an independent predictive biomarker for response to NVP-BEZ235. AR induction could add benefit during NVP-BEZ235 treatment in patients, especially with AR(+)/ER(+) breast carcinomas.

Cowden syndrome-related mutations in PTEN associate with enhanced proteasome activity.

Germline mutations in PTEN have been described in a spectrum of syndromes that are collectively known as PTEN hamartoma tumor syndrome (PHTS). In addition to being mutated in the germline in PHTS, somatic loss-of-function PTEN mutations are seen in a wide range of sporadic human tumors. Here, we show evidence of upregulated proteasome activity in PHTS-derived lymphoblasts, Pten knock-in mice and cell lines expressing missense and nonsense PTEN mutations. Notably, elevated nuclear proteasome activity occurred in cells expressing the nuclear mislocalized PTEN-K62R mutant, whereas elevated cytosolic proteasome activity was observed in cells expressing the cytosolic-predominant mutant PTEN (M3M4 and C136R). Treatment with proteasome inhibitor MG-132 was able to restore both nonsense and missense mutant PTEN protein levels in vitro. PHTS patients with destabilizing PTEN mutations and proteasome hyperactivity are more susceptible to develop neurologic symptoms such as mental retardation and autism than mutation-positive patients with normal proteasome activity. A detailed molecular and functional analysis shows that PTEN mutants most likely cause proteasome hyperactivity via 2 different mechanisms, namely, induction of proteotoxic stress and loss of protein phosphatase activity. These results provide novel insights into the cellular functions of PTEN and reveal molecular mechanisms whereby PTEN mutations increase proteasome activity and lead to neurologic phenotypes.

Utility of PTEN protein dosage in predicting for underlying germline PTEN mutations among patients presenting with thyroid cancer and Cowden-like phenotypes.

CONTEXT: Thyroid cancer is a major component of Cowden syndrome (CS). CS patients with an underlying PTEN mutation (PTEN(mut+)) have a 70-fold increased risk of developing epithelial thyroid cancer. In contrast, less than 1% of sporadic epithelial thyroid cancer patients carry a germline PTEN mutation. Cost-efficient markers capable of shortlisting thyroid cancers for CS genetic testing would be clinically useful. OBJECTIVE: Our objective was to analyze the utility of patient blood phosphate and tensin homolog deleted on chromosome 10 (PTEN) protein levels in predicting germline PTEN mutations. DESIGN, SETTING, AND PATIENTS: We conducted a 5-yr, multicenter prospective study of 2792 CS and CS-like patients, all of whom had comprehensive PTEN analysis. Analysis of PTEN and downstream proteins by immunoblotting was performed on total protein lysates from patient-derived lymphoblast lines. We compared blood PTEN protein levels between PTEN(mut+) patients and those with variants of unknown significance or wild-type PTEN (PTEN(wt/vus)). MAIN OUTCOME MEASURES: We assessed the utility of PTEN protein levels in predicting germline PTEN mutations. RESULTS: Of 2792 CS/CS-like patients, 721 patients had thyroid cancer; 582 of them (81%) had blood PTEN protein analyzed. PTEN germline pathogenic mutations were present in 27 of 582 patients (4.6%). Ninety-six percent (26 of 27) of PTEN(mut+) patients had blood PTEN protein levels in the lowest quartile as compared with 25% (139 of 555) of PTEN(wt/vus) patients (P < 0.001). Low blood PTEN levels predicted for PTEN(mut+) cases with a 99.76% negative predictive value (95% confidence interval = 98.67-99.96) and a positive test likelihood ratio of 3.84 (95% confidence interval = 3.27-4.52). CONCLUSIONS: Our study shows that low blood PTEN protein expression could serve as a screening molecular correlate to predict for germline PTEN mutation in CS and CS-like presentations of thyroid cancer.

PTEN lipid phosphatase activity and proper subcellular localization are necessary and sufficient for down-regulating AKT phosphorylation in the nucleus in Cowden syndrome.

CONTEXT: Germline mutations in PTEN are associated with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) hamartoma tumor syndrome including Cowden syndrome (CS) and Cowden-like syndrome (CSL) that predisposes to high risks of benign and malignant tumors of thyroid and breast. OBJECTIVE: The objective of the study was to analyze the subcellular pattern of phosphorylated (P)-AKT expression in nonmedullary thyroid cancers from PTEN hamartoma tumor syndrome patients and to investigate whether the lack of PTEN in the nucleus and/or lack of proper PTEN function in the nucleus affect(s) nuclear AKT activity in CS patients. DESIGN: In all, 664 patients with CS/CSL were screened for PTEN germline mutations and nonmedullary thyroid cancers. Twenty-two patients who have both pathogenic PTEN germline mutations and nonmedullary thyroid cancers were selected. Thyroid samples from these patients were stained for PTEN and P-AKT. In our in vitro study, PTEN was knocked down or overexpressed in both thyroid cancer cells and breast cancer cells, and nuclear P-AKT was compared with the control. RESULTS: Loss of PTEN protein was found in thyroid adenomas and carcinomas from all 22 (100%) PTEN(Mut+) CS/CSL patients. AKT activation was identified in 17 of 22 (77.3%) thyroid adenoma/carcinoma specimens, and most patients (63.7%) have activated nuclear AKT. Knockdown of PTEN in cells containing wild-type PTEN enhanced nuclear P-AKT, whereas expression of wild-type PTEN, but not phosphatase-dead mutants (C124S or G129E), markedly reduced nuclear P-AKT in PTEN null cells. We also showed that in breast cancer but not thyroid cancer cells, PTEN suppresses nuclear P-AKT mainly through decreasing P-AKT nuclear translocation by reducing the PIP3/P-AKT reservoir in the cytoplasm. In thyroid cancer cells, PTEN suppresses phosphorylation of AKT already resident in the nucleus. CONCLUSIONS: PTEN is necessary and sufficient for inhibiting AKT activation in the nucleus through its intact lipid phosphatase activity and proper subcellular localization.

Germline mutations in MSR1, ASCC1, and CTHRC1 in patients with Barrett esophagus and esophageal adenocarcinoma.

CONTEXT: Barrett esophagus (BE) occurs in 1% to 10% of the general population and is believed to be the precursor of esophageal adenocarcinoma (EAC). The incidence of EAC has increased 350% in the last 3 decades without clear etiology. Finding predisposition genes may improve premorbid risk assessment, genetic counseling, and management. Genome-wide multiplatform approaches may lead to the identification of genes important in BE/EAC development. OBJECTIVE: To identify risk alleles or mutated genes associated with BE/EAC. DESIGN, SETTING, AND PATIENTS: Model-free linkage analyses of 21 concordant-affected sibling pairs with BE/EAC and 11 discordant sibling pairs (2005-2006). Significant germline genomic regions in independent prospectively accrued series of 176 white patients with BE/EAC and 200 ancestry-matched controls (2007-2010) were validated and fine mapped. Integrating data from these significant genomic regions with somatic gene expression data from 19 BE/EAC tissues yielded 12 "priority" candidate genes for mutation analysis (2010). Genes that showed mutations in cases but not in controls were further screened in an independent prospectively accrued validation series of 58 cases (2010). MAIN OUTCOME MEASURES: Identification of germline mutations in genes associated with BE/EAC cases. Functional interrogation of the most commonly mutated gene. RESULTS: Three major genes, MSR1, ASCC1, and CTHRC1 were associated with BE/EAC (all P < .001). In addition, 13 patients (11.2%) with BE/EAC carried germline mutations in MSR1, ASCC1, or CTHRC1. MSR1 was the most frequently mutated, with 8 of 116 (proportion, 0.069; 95% confidence interval [CI], 0.030-0.130; P < .001) cases with c.877C>T (p.R293X). An independent validation series confirmed germline MSR1 mutations in 2 of 58 cases (proportion, 0.035; 95% CI, 0.004-0.120; P = .09). MSR1 mutation resulted in CCND1 up-regulation in peripheral-protein lysate. Immunohistochemistry of BE tissues in MSR1-mutation carriers showed increased nuclear expression of CCND1. CONCLUSION: MSR1 was significantly associated with the presence of BE/EAC in derivation and validation samples, although it was only present in a small percentage of the cases.

Naturally occurring germline and tumor-associated mutations within the ATP-binding motifs of PTEN lead to oxidative damage of DNA associated with decreased nuclear p53.

Somatic and germline mutations in PTEN (phosphatase and tensin homolog deleted on chromosome 10) are found in sporadic cancers and Cowden syndrome patients, respectively. Recent identification of naturally occurring cancer and germline mutations within the ATP-binding motifs of PTEN (heretofore referred to as PTEN ATP-binding mutations) has revealed that these mutations disrupted the subcellular localization and tumor-suppressor activity of PTEN. However, very little is known about the underlying mechanisms of PTEN ATP-binding mutations in tumorigenesis. Here we show that these mutations impair PTEN's function both qualitatively and quantitatively. On the one hand, PTEN ATP-binding mutants lose their phosphatase activity and the effect of downregulation of cyclin D1. On the other, the mislocalized mutant PTEN results in a significantly decreased nuclear p53 protein level and transcriptional activity, enhanced production of reactive oxygen species, induction of Cu/Zn superoxide dismutase as well as dramatically increased DNA double-strand breaks (DSBs). When compared with wild-type PTEN, the ATP-binding mutant PTEN has reduced half-life in vitro and decreased protein expression levels in vivo. Our data, thus, reveal a novel mechanism of tumorigenesis in patients with germline or somatic mutations affecting PTEN ATP-binding motifs, i.e. qualitative and quantitative impairment of PTEN due to the loss of its phosphatase activity, and nuclear mislocalization, resulting in rapid PTEN protein degradation, suppression of p53-mediated transcriptional activity, loss of protection against oxidative stress as well as accumulation of spontaneous DNA DSBs.

Resveratrol regulates the PTEN/AKT pathway through androgen receptor-dependent and -independent mechanisms in prostate cancer cell lines.

The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

Disruption of transforming growth factor-beta signaling by five frequently methylated genes leads to head and neck squamous cell carcinoma pathogenesis.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with low survival rates in advanced stages. To facilitate timely diagnosis and improve outcome, early detection markers (e.g., DNA methylation) are crucial for timely cancer diagnosis. In a recent publication, an epigenome-wide screen revealed a set of genes that are commonly methylated and downregulated in head and neck cancers (SEPT9, SLC5A8, FUSSEL18, EBF3, and IRX1). Interestingly, these candidates are potentially involved in the transforming growth factor-beta (TGF-beta) signaling pathway, which is often disrupted in HNSCC. Therefore, we sought to determine coordinated epigenetic silencing of these candidate genes in HNSCC as potential key disruptors of TGF-beta signaling, which could ultimately result in HNSCC progression. Through immunoprecipitation studies, all five of the investigated candidate genes were found to interact with components of the TGF-beta pathway. Overexpression of SLC5A8, EBF3, and IRX1 resulted in decreased mitotic activity and increased apoptosis. In addition, EBF3 was found to increase p21 promoter activity, and SMAD2 significantly increased IRX1 promoter activity. These findings are significant because they reveal a set of genes that interact with components of the TGF-beta pathway, and their silencing via methylation in HNSCC results in coordinated decrease in apoptosis, increased proliferation, and decreased differentiation.

AP-2alpha induces epigenetic silencing of tumor suppressive genes and microsatellite instability in head and neck squamous cell carcinoma.

BACKGROUND: Activator protein 2 alpha (AP-2alpha) is involved in a variety of physiological processes. Increased AP-2alpha expression correlates with progression in various squamous cell carcinomas, and a recent publication found AP-2alpha to be overexpressed in approximately 70% of Head and Neck Squamous Cell Carcinoma (HNSCC) patient samples. It was found to repress transcription of the tumor suppressor gene C/CAAT Enhancer Binding Protein alpha (C/EBPalpha), and its binding site correlated with upstream methylation of the C/EBPalpha promoter. Therefore, we investigated the potential for AP-2alpha to target methylation to additional genes that would be relevant to HNSCC pathogenesis. PRINCIPAL FINDINGS: Stable downregulation of AP-2alpha stable by shRNA in HNSCC cell lines correlated with decreased methylation of its target genes' regulatory regions. Furthermore, methylation of MLH1 in HNSCC with and without AP-2alpha downregulation revealed a correlation with microsatellite instability (MSI). ChIP analysis was used to confirm binding of AP-2alpha and HDAC1/2 to the targets. The effects of HDAC inhibition was assessed using Trichostatin A in a HNSCC cell line, which revealed that AP-2alpha targets methylation through HDAC recruitment. CONCLUSIONS: These findings are significant because they suggest AP-2alpha plays a role not only in epigenetic silencing, but also in genomic instability. This intensifies the potential level of regulation AP-2alpha has through transcriptional regulation. Furthermore, these findings have the potential to revolutionize the field of HNSCC therapy, and more generally the field of epigenetic therapy, by targeting a single gene that is involved in the malignant transformation via disrupting DNA repair and cell cycle control.